This project concerns the investigation of the detailed prosthetic group structure of chlorin and saturated porphyrin-containing proteins and enzymes. In the previous grant period, we established characteristic and definitive infrared (IR) and resonance Raman (RR) spectral properties of metallochlorins that are consistently diagnostic for the chlorin macrocycle in biological and model systems. In the current proposal we propose to extend those studies to the following systems: 1) The green heme d terminal oxidase from E. coli is proposed to be either a diol chlorin or a lactone chlorin. We will examine the RR spectra of the purified E. coli heme d complex in its various spin- and oxidation-states in comparison with diol and lactone substituted model complexes to determine the in vivo structure of the heme d macrocycle. 2) The green form of myoglobin (Mb) induced by exposure to sulfur compounds, sulfMb, has a sulfur-modified iron chlorin prosthetic group. The mammalian enzymes lactoperoxidase (LPO) and thyroid peroxidase (TPO) are inhibited by sulfur drugs known as goitrogens, in a reaction similar to that which leads to the formation of sulfMb. We propose to determine whether the prosthetic group of sulfLPO is indeed a sulf-chlorin, and elucidate the structural origin of the observed UV-vis spectral differences between sulfLPO and sulfMb. We will also extend the studies on sulfchlorin systems to include the stable, extractable sulfhemes from sulfMb, and if possible sulfLPO, as well as model sulfchlorin complexes. 3) We will purify the cytochrome cd1 bacterial nitrite reductase from Ps. aeruginosa and isolate the green heme d1 prosthetic group to examine spectral characteristics of dioxoisobacteriochlorins (porphyrindione). We will compare the properties of the purified cd1 enzyme and its isolated d1 prosthetic group with those of a variety of positional and substitutional isomers of oxo-chlorin and dioxo-iBC model complexes. 4) We plan to extend these studies to the analysis of sirohemes and more highly saturated porphyrins, e.g., tetrahydroporphyrins and hexahydroporphyrins, to establish spectral properties which are specific for each level of saturated porphyrin. 5) Myoglobin and other heme proteins will be reconstituted with iron chlorin complexes. We will examine the spectral properties of these chlorin-proteins in all accessible spin- and oxidation-states, as well as ligand complexes, in comparison with the protein-free model complexes.